Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance inTrypanosoma brucei

Abstract

34Defining mode of action is vital for both developing new drugs and predicting 35 potential resistance mechanisms. African trypanosome pentamidine and 36 melarsoprol sensitivity is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a 37 channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar 38 pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both 39 drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; 40 that TbAQP2 mediates pentamidine translocation or via binding to TbAQP2, with 41 subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. 42We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated 43 and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin 44 conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization 45 efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also 46 leads to impaired oligomerisation, mislocalisation, and increased turnover. These data 47suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards 48 emergence of drug resistance. 49 50 Human African trypanosomiasis (HAT) is a neglected tropical disease affecting 52 sub-Saharan countries [1-4]. HAT progresses by two stages: a haemolymphatic 53 stage, in which the parasite successfully colonises the bloodstream, lymphatics, skin, 54 adipose tissue and organs and a meningoencephalic stage characterised by the 55 emergence of parasites in the central nervous system (CNS) [2,5]. Several drugs are 56 used to treat HAT; currently suramin and pentamidine are the drugs of choice for 57 treatment of the haemolymphatic stage of T. brucei rhodesiense and T. brucei 58 gambiense infections respectively, whereas melarsoprol, eflornithine or combined 59 nifurtimox-eflornithine (NECT) therapy are recommended for the meningoencephalic 60 stage [6,7]. 61 Two new drugs, fexinidazole and acoziborole, recently completed clinical trials 62 and opened a new front in HAT chemotherapy [8,9]. Drug development, successful 63 public health initiatives and active case-monitoring programs have all contributed to 64 the anticipated eradication of gambiense HAT as a major public health problem in the 65 coming decade [10]. However, vigilance and understanding of drug mechanisms and 66 possible resistance pathways remain essential to maintain this situation, and 67rhodesiense HAT cannot be eliminated in this way as it is highly zoonotic [11]. 68Genome-wide RNAi screens identified a number of genes associated with 69 pentamidine sensitivity that, together with evidence from melarsoprol-pentamidine 70 cross-resistance (MPXR), identified aquaglyceroporin 2 as the primary determinant for 71 drug-uptake [12,13], alongside lesser roles for the TbAT1/P2 aminopurine transporter 72 and the Low Affinity Pentamidine transporter LAPT1 [14]. 73 Aquaglyceroporins (AQPs) are an ancient family of multi-pass membrane 74 proteins, containing b...

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins

“…Trypanosoma brucei AQP1 and AQP3 display the internal arrangements in the protein pore observed in canonical AQPs, including the canonical ‘NPA’ within two half α -helices and a narrower ‘aromatic/arginine’ (ar/R) motif (Beitz, 2005). Interestingly, TbAQP2 does not retain this canonical configuration, displaying an unconventional ‘NPS/NSA’ filter motif and rearrangement in the ar/R motif that is replaced by a neutral leucine at position 264 (L264), followed by aliphatic, rather than aromatic, residues (A88, I110, V249 and L258), which are equivalent to the ‘IVLL’ motif observed in the selectivity pore of canonical AQPs (de Groot and Grubmuller, 2001; Baker et al ., 2013; Quintana et al ., 2020). These structural features indicate that TbAQP2 can accommodate larger solutes through the selectivity pore (Uzcategui et al ., 2004).…”

“…Further biochemical and genetic manipulation studies demonstrated that deletion of AQP2, but not AQP3, led to a significant increase in the EC 50 of both compounds, mirroring the behaviour observed in previously generated laboratory strains and field isolates (Munday et al ., 2014; Graf et al ., 2015 b ; Song et al ., 2016). Other observations such as localization to the flagellar pocket in the bloodstream form (Munday et al ., 2014; Graf et al ., 2015 b ; Song et al ., 2016; Quintana et al ., 2020), as well as the unusual pore structure discussed above, led to the hypothesis that pentamidine and melarsoprol are likely to interact with high affinity to AQP2 located in the flagellar pocket (Alghamdi et al ., 2020), posing the question of how these compounds are internalized and also the mechanisms for resistance.…”

“…In the endocytosis model, ubiquitination of TbAQP2 at the flagellar pocket is central for subsequent ubiquitination-mediated intracellular trafficking and delivery to intracellular organelles such as the lysosome. Indeed, TbAQP2 forms a stable homomultimeric complex in the flagellar pocket where ubiquitination is likely to take place on individual monomers (Quintana et al ., 2020).…”

“…This was demonstrated by TbAQP3 mutants containing the amino acids of the selectivity pore from TbAQP2 possessing increased capacity for pentamidine uptake (Alghamdi et al ., 2020). Moreover, pentamidine permeation through TbAQP2 seems to be further aided by the intrinsic membrane potential and is not abrogated by partially blocking endocytic uptake (Alghamdi et al ., 2020; Quintana et al ., 2020), albeit at a rate that is considerably slower than for lower molecular weight solutes, which in essence implies a leak in the AQP2 permeability barrier.…”

“…Mutations on these residues profoundly impair protein stability, rendering the parasites resistant to pentamidine and melarsoprol. (Quintana et al ., 2020). (C) Number of clear AQP paralogues detected in representative taxa.…”